Use of pedf-derived polypeptides for promoting stem cells proliferation and wound healing

ABSTRACT

Disclosed herein is a synthetic peptide, which has an amino acid sequence that has 20-39 amino acid residues. The synthetic peptide has at least 80% amino acid sequence identity to SEQ ID NO: 1, and includes at least 20 consecutive residues that has at least 90% amino acid sequence identity to residues 11-30 of SEQ ID NO: 1. Also disclosed herein are compositions containing the synthetic peptide and applications thereof. According to various embodiments of the present disclosure, the synthetic peptide is useful in promoting stem cells proliferation or wound healing.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 13/428,996, filed Mar. 23, 2013, which claims priority toTaiwan application NO: 100109945, filed Mar. 23, 2011, the entireties ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure relates to stem cell proliferation. Moreparticularly, the disclosed invention relates to the proliferation oflimbal epithelial stem cells (LSCs) or hair follicle stem cells (HFSCs).

2. Description of Related Art

Stem cells are associated with the formation, repair, and maintenance oftissues during embryogenesis and injuries and aging in the adults. Stemcells are unique in their self-renewal capacity and differentiationpotential. The capacity of self-renewal enables these stem cells to makemore copies thereof, whereas differentiation potential allows them todifferentiate into multiple or all the germ lineages. These propertiesare collectively defined as the “stemness” of stem cells.

Mammalian stems cells are broadly categorized into embryonic stem cellsand adult stem cells. Embryonic stem cells are derived from the innercell mass of blastocysts which is formed in the early embryogenesis ofmammals. These embryonic stem cells are totipotent, which means they maygive rise to all the tissues of a complete organism. Embryonic stemcells can be maintained in culture as undifferentiated cell lines orinduced to differentiate into many different lineages, and therefore,they have been widely used in fields of molecular biology and medicine.However, one drawback of maintaining embryonic stem cells in culture istheir tendency to differentiate spontaneously and thereby lose theirproliferation capacity over time. Many tissues in adult animals havebeen shown to contain reservoirs of stem cells, which are called “adultstem cells.” Adult stem cells, in comparison with to embryonic stemcells, have a more restricted differentiation capacity, and are usuallylineage-specific. These adult stems cells are often associated with therepair and maintenance of tissues. For example, bone marrow stem cellsmay migrate to various tissues after injuries, while tissue stem cellslocated outside the bone marrow may repair the tissues in which theyreside.

Limbus anatomically locates between cornea and sclera at ocular surface.The basal layer of limbal epithelium is enriched with a special cellpopulation, named limbal epithelial stem cells (LSCs). The cornea is astratified squamous epithelium with a rapid turnover property thatmaintains corneal transparency and visual acuity. The renewal of cornealepithelium is supported by the transient amplifying cells (TACs) who aregenerated by asymmetric division of LSCs. In addition, LSCs are normallyslow cycling cells, but activated by corneal wounding that enablecorneal damage repaired.

Partial or total loss or dysfunction of LSCs (clinical termed LSCdeficiency) leads to corneal neovascularization, recurrent erosions,stromal scarring, ulceration, thereby causing vision loss. LSCdeficiency may arise following injuries including chemical or thermalburns and through diseases such as aniridia, chronic infection (e.g.,trachoma), mycotic keratitis, and Stevens Johnson syndrome. Currently,transplantation of the ex vivo expanded limbal epithelial sheet hasbecome the most widely used therapy for LSC deficiency. This therapeuticapproach generally involves placing a small limbal biopsy removed fromeither the patient or a donor on transplantable carriers such as denudedhuman amniotic membrane to support limbal cells migrating out from thebiopsy and outgrowth to form a limbal-like epithelial sheet. Inaddition, enzymatically isolating the limbal cells from limbal tissueand their expansion by suspension culture systems contributes todecrease the LSCs spontaneously differentiation. The failure of limbaltransplantation is often arising from the depletion of LSCs in culture.Accordingly, it is desirable to effectively expand the quantity of LSCsin vitro or ex vivo before the transplantation.

Adult mammalian epidermis consists of the hair follicles (HFs), thesebaceous glands (SGs), and the interfollicular epidermis. Thehomeostasis of each of these three epithelial compartments is supportedby their own stem cell (SC) population. Hair follicle stem cells (HFSCs)reside in the hair follicle bulge; they are multipotent and have thecapacity to give rise to all epidermal lineages. HFSCs are predominantlyresponsible for reconstituted HFs during homeostasis rather thanresponsible for the formation of epidermis. However, after skinwounding, HFSCs contribute to interfollicular epidermal repair. Yet, thein vivo proliferation of HFSCs following the injuries is not fastenough, and may slowdown the wound healing process. Moreover, in theevent of large amount of skin loss, such as those resulted frominfection, surgical excision, and burn, there may be insufficientavailable HFSCs to participate in wound healing.

In view of the foregoing, there exists a need in the art to provide amethod of promoting the proliferation of stem cells, which in turn maypromote wound healing.

SUMMARY

The following presents a simplified summary of the disclosure in orderto provide a basic understanding to the reader. This summary is not anextensive overview of the disclosure and it does not identifykey/critical elements of the present invention or delineate the scope ofthe present invention. Its sole purpose is to present some conceptsdisclosed herein in a simplified form as a prelude to the more detaileddescription that is presented later.

The present disclosure is based, at least, on the finding that syntheticpeptides derived from pigment epithelium-derived factor (PEDF) maypromote the proliferation of limbal epithelial stem cells or hairfollicle stem cells, and thereby promote wound healing processesrespectively associated with these two types of stem cells. ThePEDF-derived synthetic peptides of this invention are, therefore, usefulas an agent or a medicament for treating wounds.

Accordingly, in one aspect, the present disclosure is directed to asynthetic peptide for promoting stem cells proliferation.

According to embodiments of the present disclosure, the syntheticpeptide is 20-39 amino acid residues in length, and has an amino acidsequence that is at least 80% identical to SEQ ID NO: 1. Also, the aminoacid sequence comprises at least 20 consecutive residues, which is atleast 90% identical to residues 11-30 of SEQ ID NO: 1, such that thesynthetic peptide is useful in promoting the proliferation of limbalepithelial stem cells or hair follicle stem cells. Non-limiting examplesof such synthetic peptides include those having an amino acid sequenceof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,or SEQ ID NO: 8.

In another aspect, the present disclosure is directed to a compositionfor promoting stem cells proliferation. This composition is suitable forpromoting the in vivo, ex vivo, or in vitro proliferation of stem cells,in particular, limbal epithelial stem cells and hair follicle stemcells.

According to one embodiment of the present disclosure, the compositioncomprises a synthetic peptide according to any of the above-mentionedaspect/embodiments, and the synthetic peptide is present in an effectiveamount sufficient to promote the stem cell proliferation. Thecomposition also comprises a carrier for the synthetic peptide. In thecase of in vitro proliferation, the carrier may be a powdered culturemedium suitable for culturing the stem cells. According to embodimentsof the present disclosure, the synthetic peptide is present in theculture medium in an amount of about 1-100 nM, and preferably, about25-50 nM. In the case of in vivo proliferation, the carrier may be apharmaceutically acceptable carrier suitable for administering to aliving mammal, including human. For example, the pharmaceuticallyacceptable carrier may be any of a liquid, gel, cream, ointment,adhesive, amniotic membrane, skin substitute, artificial skin, or skinequivalents.

In yet another aspect, the present invention is directed to a method forpromoting stem cells proliferation.

According to one embodiment, the method comprises treating the stemcells with an effective amount of the synthetic peptide according to anyof the above-mentioned aspect/embodiments, thereby promoting the stemcells to proliferate. In various embodiments, the stem cells areproliferated in vivo, ex vivo, or in vitro.

In still another aspect, the present invention is directed to a methodfor promoting healing of a corneal or an epithelial wound in a subject.The subject may be any animal classified as a mammal, including human.

In one embodiment, the method comprises administering to the subject atherapeutically effective amount of the composition according to theabove-mentioned aspect/embodiments of the present disclosure so as topromote stem cells associated with the corneal or epithelial wound ofthe subject to proliferate. In practice, the composition may beadministered via topical administration, subconjunctival injection,subcutaneous injection, or intradermal injection.

According to some embodiments, the corneal wound is caused by pterygium;recurrent corneal erosion; limbal deficiency caused by dry eye or drugtoxicity; corneal damage caused by chemical or thermal burn, or herpesvirus; keratopathy induced by contact lens or radiation; corneal lesionsinduced by Stevens Johnson Syndrome, aniridia, limbal tumors, ocularcicatricial pemphigoid (OCP), limbal deficiency-induced cornealneovascularization, or diabetes-induced difficulty of corneal woundhealing.

According to other embodiments, the epithelial wound is caused bysurgical excision, skin ulcer derived from infection, chemical orthermal burn, donor site of full thickness and split thickness skingraft, bedsore, and ischemic necrosis, or diabetes-induced andage-induced difficulty of skin surface wound healing.

Many of the attendant features and advantages of the present disclosurewill becomes better understood with reference to the following detaileddescription considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The present description will be better understood from the followingdetailed description read in light of the accompanying drawings, where:

FIG. 1A provides immunofluorescence microscopy photographs and FIG. 1Bis a histogram, and together, they illustrate the in vitro proliferationof LSCs from treatment groups according to one example of the presentdisclosure. LSC proliferation was determined by BrdU labeling for 2hours. LSCs (ΔNp63α, green) and BrdU (red) were detected byimmunofluorescence microscopy (original magnification, ×400). Tenrandomly selected fields in each group were photographed, and thepercentage of BrdU and ΔNp63α-double positive cells (pale pink) pertotal ΔNp63α-positive cells was calculated. *P<0.002 versus untreatedcells.

FIG. 2A provides photographs and FIG. 2B is a histogram, and together,they illustrate the corneal wound-healing of mice from treatment groupsaccording to another example of the present disclosure. Mice eyes werestained with topical fluorescein after formation of a 2 mm corneal woundfollowed for 48 hours and then photographs of the fluorescein-stainedcorneas were captured with a CCD camera (Olympus). Nine groups of micewere included: controls receiving 20 μl of eye ointment mixed with DMSO(less than 0.05 μL) and mice given 20 μl of a short peptide (50μM)-mixed eye ointment once a day. Wound area was measured by AdobePhotoshop CS3 10.0. Histogram of residual epithelial defect (%) in micecorneas is presented as percentage of the original wound. Significantlydifferent from control groups at 24 hours and 48 hours. *P<0.05 versuscontrol group.

FIG. 3A provides immunofluorescence microscopy photographs and FIG. 3Bis a histogram, and together, they illustrate the in vivo limbusproliferation at 24 hours post-corneal injury according to the previousexample of the present disclosure. Double immunofluorescence of sectionsof control ointment and PEDF-derived short peptides (29-mer, 20-mer and18-mer) ointment-treated eyes stained with BrdU- and ΔNp63α-specificantibodies. ΔNp63α is expressed in the cell nucleus as confirmed byHoechst 33258 counterstaining. P<0.005 versus control eye.

FIG. 4A provides histologic photographs (left panels, Hematoxylin andEosin (H&E) staining, magnification, ×400), immunofluorescencemicroscopy photographs (middle panels, magnification, ×1000), and mergedphotographs (right panels); together, they illustrate layers ofstratified limbal epithelium of mice at day 5 post-corneal injuryaccording to the previous example of the present disclosure.Immunofluorescence analysis showed the distribution and level ofΔNp63α-positive cells at limbus. Normal indicates uninjured eye. Corneaof normal eye that has no ΔNp63α-positive cell acted as ΔNp63α negativeimmunostaining control. FIG. 4B is a histogram illustrating levels ofΔNp63α-positive cells at limbus. Results of immunofluorescence wereevaluated from 6 sections per mouse cornea, and 6 mice at eachtreatment. A labeling index was computed as the number of labeled cellsdivided by the total number of cells observed by a Hoechst 33258fluorescent nuclear stain. P<0.005 versus control eye.

FIG. 5A provides immunofluorescence microscopy photographs and FIG. 5Bis a histogram, and together, they illustrate the in vitro proliferationof HFSCs from treatment groups according to one example of the presentdisclosure. HFSC proliferation was determined by BrdU labeling for 2hours. HFSCs (Lgr6, green) and BrdU (red) were detected byimmunofluorescence microscopy (original magnification, ×1000). Twentyrandomly selected fields in each group were photographed, and thepercentage of BrdU and Lgr6-double positive cells (pale pink) per totalLgr6-positive cells was calculated. *P<0.001 versus untreated cells.

FIG. 6A provides photographs and FIG. 6B is a histogram, and together,they illustrate the skin (epithelial) wound-healing of mice fromtreatment groups according to another example of the present disclosure.4 mm punch wounds were made on the dorsal side of C57/B6 mice. Skinointment was treated once a day. Photographs of the wounded skin andruler (scale 10 mm) were captured with a CCD camera (Olympus). Theresidual epithelial defect (%) is presented as percentage of theoriginal wound. Data are shown as mean±SE. Significantly different fromcontrol groups are post-wounding for 4 and 7 days (*P<0.05).

FIGS. 7A to 7E provide quantification of wound healing at day 4. FIG. 7Aprovides representative photographs from skin specimen stained by Massontrichrome (original magnification, ×100) and the 29-mer and 20-mertreated wound showed better wound healing. Arrowheads (▾) and arrows (↓)denote initial wound margins and the foremost tips of thehyperproliferative epithelium (HE; red color), respectively. Data arerepresentative of 10-16 wounds (2 wounds per mouse) in each group.*P<0.02 versus control wound. FIG. 7B is a histogram illustrating theresidual epithelial defect (%) in mice from each treatment group; onlysections of the middle of the wounds were used for quantification. Dataare shown as mean±SE. FIG. 7C provides representative photographs ofwound edge. GT: granulation tissue (blue color); Es: eschar (deep-red).29-mer treated wound showed thicker HE. FIGS. 7D and 7E are histogramsproviding quantifications of the areas of HE and GT using AdobePhotoshop CS3 10.0. *P<0.02 versus control wound.

FIGS. 8A and 8B provide quantification of wound healing at day 7.

FIG. 8A provides representative photographs from skin specimen stainedby Masson trichrome (original magnification, ×100) and the 29-mer, Mo29-mer, and 20-mer treated wound showed better wound healing. Arrowheads(▾) and arrows (↓) denote initial wound margins and the foremost tips ofthe hyperproliferative epithelium, respectively. FIG. 8B is a histogramillustrating the residual epithelial defect (%) in mice from eachtreatment group; only sections of the middle of the wounds were used forquantification. Data are shown as mean±SE. *P<0.05 versus control wound.

FIGS. 9A and 9B illustrate histological analysis of cell replication atday 4 post-skin wounding. FIG. 9A provides photographs in whichspecimens were immunehistologically stained with BrdU to observe DNAreplication (deep brown color) and counterstained with hematoxylin toobserve nuclei. Representative photograph showed cell replication mainlyat basal layer of HE tissue as indicated by arrow (↓). (Originalmagnification, ×200). FIG. 9B is a histogram illustrating numbers ofBrdU-positive cells at basal layer of HE tissue. A labeling index (%)was computed as the number of labeled cells divided by the total numberof cells at basal layer of HE tissue. *P<0.05 versus control wound.

FIGS. 10A and 10B illustrate histological analysis of the distributionof HFSCs at day 4 post-skin wounding. FIG. 10A provides photographs inwhich specimens were immunehistologically stained with Lgr6 to observeHFSC-derived cells at HE tissue and counterstained with hematoxylin toobserve nuclei. Original magnification, ×400. FIG. 10B presentsphotographs in which sections of 20-mer ointment-treated wound werestained with BrdU- and Lgr6-specific antibodies. Lgr6 is expressed inthe cell nucleus as confirmed by Hoechst 33258 counterstaining.

DESCRIPTION

The detailed description provided below in connection with the appendeddrawings is intended as a description of the present examples and is notintended to represent the only forms in which the present example may beconstructed or utilized. The description sets forth the functions of theexample and the sequence of steps for constructing and operating theexample. However, the same or equivalent functions and sequences may beaccomplished by different examples.

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless otherwise defined herein, scientific andtechnical terminologies employed in the present disclosure shall havethe meanings that are commonly understood and used by one of ordinaryskill in the related art. Unless otherwise required by context, it willbe understood that singular terms shall include plural forms of the sameand plural terms shall include the singular. Specifically, as usedherein and in the claims, the singular forms “a” and “an” include theplural reference unless the context clearly indicates otherwise.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in therespective testing measurements. Also, as used herein, the term “about”generally means within 10%, 5%, 1%, or 0.5% of a given value or range.Alternatively, the term “about” means within an acceptable standarderror of the mean when considered by one of ordinary skill in the art.Other than in the operating/working examples, or unless otherwiseexpressly specified, all of the numerical ranges, amounts, values andpercentages such as those for quantities of materials, durations oftimes, temperatures, operating conditions, ratios of amounts, and thelikes thereof disclosed herein should be understood as modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the present disclosureand attached claims are approximations that can vary as desired. At thevery least, each numerical parameter should at least be construed inlight of the number of reported significant digits and by applyingordinary rounding techniques.

As used herein, the term “peptide” denotes a polymer of amino acidresidues. By the term “synthetic peptide” as used herein, it is meant apeptide which does not comprise an entire naturally occurring proteinmolecule. The peptide is “synthetic” in that it may be produced by humanintervention using such techniques as chemical synthesis, recombinantgenetic techniques, or fragmentation of whole antigen or the like.Throughout the present disclosure, the positions of any specified aminoacid residues within a peptide are numbered starting from the N terminusof the peptide.

The term “stem cell” as used herein, refers to a cell that retains thecapacity, under certain circumstances, to proliferate withoutsubstantially differentiating; as well as the capacity or potential,under particular circumstances, to differentiate to a more specializedor differentiated phenotype. As discussed hereinabove, the presentdisclosure is particularly related to two adult tissue stem cells, i.e.,limbal epithelial stem cells and hair follicle stem cells. Tissue stemcells are located in sites called niches, which differ among varioustissues. For example, limbal epithelial stem cells reside preferentiallyin the basal layer of peripheral cornea in the limbal zone, whereas hairfollicle stem cells reside in the hair follicle bulge.

As used herein, “proliferating” and “proliferation” refers to anincrease in the number of cells in a population by means of celldivision.

The term “promote” or “promoting” is meant to refer to a positivealteration; in particular a statistically significant positivealteration. The positive alteration means an increase of at least 10% ascompared to a reference level.

“Percentage (%) amino acid sequence identity” with respect to thesynthetic polypeptide sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the specific polypeptidesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percentage sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. For purposes herein,sequence comparison between two amino acid sequences was carried out bycomputer program Blastp (protein-protein BLAST) provided online byNation Center for Biotechnology Information (NCBI). The percentage aminoacid sequence identity of a given amino acid sequence A to a given aminoacid sequence B (which can alternatively be phrased as a given aminoacid sequence A that has a certain % amino acid sequence identity to agiven amino acid sequence B) is calculated by the formula as follows:

$\frac{X}{Y} \times 100\%$

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program BLAST in that program's alignment of Aand B, and where Y is the total number of amino acid residues in A or B,whichever is shorter.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation. The carrier can be in theform of a solid, semi-solid, or liquid diluent, cream or a capsule.

As used herein, the terms “treat” or “treating” or “treatment” refer topreventative (e.g., prophylactic), curative or palliative treatment. Theterm “treating” as used herein refers to application or administrationof the composition of the present disclosure to a subject, who has amedical condition, a symptom of the condition, a disease or disordersecondary to the condition, or a predisposition toward the condition,with the purpose to partially or completely alleviate, ameliorate,relieve, delay onset of, inhibit progression of, reduce severity of,and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease,disorder, and/or condition and/or to a subject who exhibits only earlysigns of a disease, disorder, and/or condition for the purpose ofdecreasing the risk of developing pathology associated with the disease,disorder, and/or condition. Treatment is generally “effective” if one ormore symptoms or clinical markers are reduced as that term is definedherein. Alternatively, a treatment is “effective” if the progression ofa disease is reduced or halted. That is, “treatment” includes not justthe improvement of symptoms or decrease of markers of the disease, butalso a cessation or slowing of progress or worsening of a symptom thatwould be expected in absence of treatment. Beneficial or desiredclinical results include, but are not limited to, alleviation of one ormore symptom(s), diminishment of extent of disease, stabilized (i.e.,not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable.

The term “effective amount” as used herein refers to the quantity of acomponent which is sufficient to yield a desired response. The term“therapeutically effective amount” as used herein refers to the amountof therapeutically agent of pharmaceutical composition to result in adesired “effective treatment” as defined hereinabove. The specifictherapeutically effective amount will vary with such factors as theparticular condition being treated, the physical condition of thepatient (e.g., the patient's body mass, age, or gender), the type ofmammal or animal being treated, the duration of the treatment, thenature of concurrent therapy (if any), and the specific formulationsemployed. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the compound or composition areoutweighed by the therapeutically beneficial effects.

The term “subject” refers to a mammal including the human species thatis treatable with the compositions and/or methods of the presentinvention. The term “subject” is intended to refer to both the male andfemale gender unless one gender is specifically indicated.

Pigment epithelium-derived factor (PEDF) is a multifunctional secretedprotein that has anti-angiogenic, anti-tumorigenic, and neurotrophicfunctions. Human PEDF protein is a secreted protein of roughly 50 kDasize and 418 amino acids in length. A 34-mer fragment (residues 44-77)and a 44-mer fragment (residues 78-121) of PEDF have been identified tohave anti-angiogenic and neurotrophic properties, respectively.

The full-length human PEDF molecule has been previously reported as afactor for enhancing symmetrical self-renewal of neural stem cells (USPatent Publication NO: 2010/0047212 by Gomez et al.). Said applicationalso disclosed a C-ter PEDF (amino acids 195-400) which was unable topromote self-renewal of neural stem cells. Their results implied that adomain within the first 194 N-terminal amino acids may be necessary forself-renewal of neural stem cells; however, no specific N-terminalfragments were described or tested. Gomez et al. further taught that theC-ter PEDF was capable of competitively inhibiting the self-renewaleffect of the full length PEDF on neural stem cells, which implied thatthe C-ter PEDF must also play a role in the self-renewal activity of thenative PEDF. Nevertheless, Gomez et al. did not explicitly or implicitlytaught or suggested any specific PEDF fragment that is capable ofenhancing symmetrical self-renewal of neural stem cells. In addition, noeffects of PEDF (full-length or fragment thereof) on other types orsources of stem cell, other than neural stem cells, have been disclosedor tested. Given the diversity of conditions of the niches in whichvarious tissue stem cells reside and the complexity of the inducing andregulation of symmetrical and asymmetrical stem cell proliferations,Gomez et al. provided no direction or guidance regarding stem cell typesthat may be promoted to proliferate under the action of the full-lengthPEDF molecule.

The present disclosure is based, at least, on the finding that syntheticpeptides derived from PEDF may promote the proliferation of limbalepithelial stem cells or hair follicle stem cells. One inventive featureof the present invention lies in that the synthetic peptides are muchshorter (39 amino acid residues at most) than the full-length PEDF andthus overcomes the limitations associated with the clinical use ofconventional protein drugs, including high manufacturing cost, lowbioavailability, and poor pharmacokinetics. Further, the presentdisclosure demonstrates that the synthetic peptides of this inventionmay be delivered transdermally, and thereby eliminates the need of aninvasive delivery means (e.g., injection) as is generally required forthe delivery of a protein drug. Also, findings disclosed in the presentinvention establish that the synthetic peptides are effective inpromoting the limbal epithelial stems cells and hair follicle stem cellsto proliferate, both in vivo and in vitro. Accordingly, the presentsynthetic peptides are useful for treating corneal and epithelialwounds.

Thus, in one aspect, the present disclosure is directed to a syntheticpeptide for promoting stem cells proliferation.

According to embodiments of the present disclosure, the syntheticpeptide is 20-39 amino acid residues in length, and has at least 80%amino acid sequence identity with the amino acid sequence ofLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGT (SEQ ID NO: 1). For example, thesynthetic peptide may have an amino acid sequence identity of about 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100 percent with SEQ ID NO: 1. Also, the synthetic peptidecomprises at least 20 consecutive residues that are at least 90%identical to residues 11-30 of SEQ ID NO: 1. Specifically, the 20consecutive amino acid residues may have about 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100 percent amino acid sequence identity withresidues 11-30 of SEQ ID NO: 1.

In one embodiment, the synthetic peptide has the sequence of SEQ ID NO:1, which is 39 amino acids in length. This synthetic peptide is alsoreferred to as 39-mer in the examples hereinbelow. This 39-mer peptideis a short variant derived from the known 44-mer of human PEDF, whichcorresponds to residues 78-121 of PEDF.

Experiments provided hereinbelow confirm that there are several short,synthetic PEDF peptides derived from the 39-mer are effective inpromoting the proliferation of limbal epithelial stem cells or hairfollicle stem cells.

For example, a 34-mer synthetic peptide having the sequence ofALSALSLGAEQRTESIIHRALYYDLISSPDIHGT (SEQ ID NO: 2) is proved to beeffective in enhancing the HFSC and LSC proliferation according to oneexample provided hereinbelow. According to the process for estimatingpercentage of sequence identity between any two given sequences providedabove, the 34-mer has a 100% amino acid sequence identity to the 39-mer,and the 6^(th)-25^(th) amino acid residues of the 34-mer has a 100%amino acid sequence identity to the amino acid residues 11-30 of the39-mer.

Additionally, a 29-mer synthetic peptide having the sequence ofSLGAEQRTESIIHRALYYDLISSPDIHGT (SEQ ID NO: 3) has been confirmed to beeffective in promoting LSC and HFSC proliferations, as well as cornealand epithelial wound-healing, according to various examples hereinbelow.This 29-mer has a 100% amino acid sequence identity to the 39-mer, andthe 1^(st)-20^(th) amino acid residues of the 29-mer has a 100% aminoacid sequence identity to the amino acid residues 11-30 of the 39-mer.

In some examples, it is established that a 20-mer is also effective inpromoting LSC and LFSC proliferations. The 20-mer has the sequence ofSLGAEQRTESIIHRALYYDL (SEQ ID NO: 5), which is completely identical tothe amino acid residues 11-30 of the 39-mer (100% amino acid sequenceidentity), and has a 100% amino acid sequence identity to the 39-mer.

Two synthetic peptides derived from mouse PEDF are also effective inpromoting LSC and LFSC proliferations and corneal and epithelialwound-healing. The first mouse-derived peptide is also referred to as Mo29-mer in the present disclosure. The Mo 29-mer has a sequence ofSLGAEHRTESVIHRALYYDLITNPDIHST (SEQ ID NO: 7), which has a 83% amino acidsequence identity to 39-mer, and the first 20 amino acid residuesthereof has a 90% amino acid sequence identity to the 11-30 amino acidresidues of the 39-mer. Another mouse-derived peptide, Mo 20-mer has asequence of SLGAEHRTESVIHRALYYDL (SEQ ID NO: 8). The Mo 20-mer has a 90%amino acid sequence identity to either the 39-mer or the 11-30 aminoacid residues of the 39-mer.

The synthetic Peptides of the invention can be synthesized by commonlyused methods such as t-BOC or FMOC protection of alpha-amino groups.Both methods involve stepwise syntheses whereby a single amino acid isadded at each step starting from the C terminus of the peptide. Peptidesof the invention can also be synthesized by the well-known solid phasepeptide synthesis methods.

Other synthetic peptides with conservative variation with respect to the39-mer are also contemplated. The term “conservative variation” as usedherein denotes the replacement of an amino acid residue by another,biologically similar residue. Examples of conservative variationsinclude the substitution of one hydrophobic residue such as isoleucine,valine, leucine or methionine for one another, or the substitution ofone polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

The synthetic peptides according to above-mentioned embodiments may beformulated into compositions for promoting stem cells proliferation,which falls within another aspect of the present disclosure. In variousembodiments of the present disclosure, this composition is suitable forpromoting the in vivo, ex vivo, or in vitro proliferation of stem cells,in particular, LSCs and HFSCs.

According to one embodiment of the present disclosure, the compositioncomprises a synthetic peptide according to any of the above-mentionedaspect/embodiments, and the synthetic peptide is present in an effectiveamount sufficient to promote the stem cell proliferation. Thecomposition also comprises a carrier for the synthetic peptide.

For in vitro proliferation, the carrier may be a powdered culture mediumsuitable for culturing the stem cells after reconstitution. Thereconstitution may be achieved by first dissolving the powdered culturemedium in pyrogen-free water, isotonic saline, or phosphate buffersolution; and followed by adjusting the pH of the re-constituted mediumto a proper range, e.g., between 6.8 to 7.4. According to embodiments ofthe present disclosure, the synthetic peptide is present in the culturemedium in an amount of about 1-100 nM, and preferably, about 25-50 nM.In working examples provided hereinbelow, the synthetic peptide ispresent at a concentration of 25 nM and 50 nM respectively in the mediafor culturing LSC and HFSC.

For in vivo proliferation, the carrier may be a pharmaceuticallyacceptable carrier suitable for administering to a living mammal,including human. For example, the pharmaceutically acceptable carriermay be any of a liquid, gel, cream, ointment, adhesive, amnioticmembrane, skin substitute, artificial skin, or skin equivalents.

Some examples of substances which can serve as pharmaceuticallyacceptable carriers are gelatin, excipients, pyrogen-free water,isotonic saline, and phosphate buffer solutions. In one embodiment, thepharmaceutically acceptable carrier comprises an ophthalmicallyacceptable pharmaceutical excipient.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with a synthetic peptide is basically determined by the waythe composition is to be administered. The composition of the presentinvention may be administered locally (e.g., topically,subconjunctivally, or intradermally) or systemically (e.g.subcutaneously).

As used herein, the term “topical administration” refers toadministration onto any accessible body surface of any human or animalspecies, preferably the human species, for example, the skin or theouter surface of the eye. Suitable pharmaceutically-acceptable carriersfor topical application include those suitable for use in liquids(including solutions and lotions), creams, gels, and the like.Advantageously, the composition is sterile and can be in dosage unitform, e.g., suitable for topical ocular use. The composition can bepackaged in a form suitable for metered application, such as incontainer equipped with a dropper.

In one embodiment, the composition is a solution prepared using aphysiological saline solution as a carrier. In another embodiment, thecomposition is an ointment containing the synthetic peptide at aconcentration of about 50 μM. The pH of the solution or ointment is,preferably, maintained between 4.5 and 8.0 using an appropriate buffersystem. A neutral pH is more preferred. Alternatively, the syntheticpeptide may be incorporated in an amniotic membrane, skin substitute,artificial skin, or other skin equivalents that are suitable to beapplied to the wound site.

For subconjunctival, intradermal or subcutaneous injection, thesynthetic peptide may be formulated with a pharmaceutically acceptablecarrier such as a sterile aqueous solution which is preferably isotonicwith the blood of the recipient. Such formulations may be prepared bydissolving or suspending the solid active ingredient in water containingphysiologically compatible substances such as sodium chloride, glycine,and the like, and having a buffered pH compatible with physiologicalconditions to produce an aqueous solution, and rendering said solutionsterile.

Compositions of the invention can also comprise various additives knownto those skilled in the art. For example, solvents, including relativelysmall amounts of alcohol, may be used to solubilize certain drugsubstances. Other optional pharmaceutically-acceptable additives includeopacifiers, antioxidants, fragrance, colorant, gelling agents,thickening agents, stabilizers, surfactants, and the like. Other agentsmay also be added, such as antimicrobial agents, to prevent spoilageupon storage, i.e., to inhibit growth of microbes such as yeasts andmolds. Permeation enhancers and/or irritation-mitigating additives mayalso be included in the composition of the present invention.

In yet another aspect, the present invention is directed to a method forpromoting stem cells proliferation.

According to one embodiment, the method comprises treating the stemcells with an effective amount of the synthetic peptide according to anyof the above-mentioned aspect/embodiments, thereby promoting the stemcells to proliferate. In various embodiments, the stem cells areproliferated in vivo, ex vivo, or in vitro.

In still another aspect, the present invention is directed to a methodfor promoting healing of a corneal or an epithelial wound in a subject.The subject may be any animal classified as a mammal, including human.

In one embodiment, the method comprises administering to the subject atherapeutically effective amount of the composition according to theabove-mentioned aspect/embodiments of the present disclosure so as topromote stem cells associated with the corneal or epithelial wound ofthe subject to proliferate. In practice, the composition may beadministered via topical administration, subconjunctival injection,subcutaneous injection, or intradermal injection.

According to some embodiments, the corneal wound is caused by pterygium;recurrent corneal erosion; limbal deficiency caused by dry eye or drugtoxicity; corneal damage caused by chemical or thermal burn, or herpesvirus; keratopathy induced by contact lens or radiation; corneal lesionsinduced by Stevens Johnson Syndrome, aniridia, limbal tumors, ocularcicatricial pemphigoid (OCP), limbal deficiency-induced cornealneovascularization, or diabetes-induced difficulty of corneal woundhealing.

According to other embodiments, the epithelial wound is caused bysurgical excision, skin ulcer derived from infection, chemical orthermal burn, donor site of full thickness and split thickness skingraft, bedsore, and ischemic necrosis, or diabetes-induced andage-induced difficulty of skin surface wound healing.

The following Examples are provided to elucidate certain aspects of thepresent invention and to aid those of skilled in the art in practicingthis invention. These Examples are in no way to be considered to limitthe scope of the invention in any manner. Without further elaboration,it is believed that one skilled in the art can, based on the descriptionherein, utilize the present invention to its fullest extent. Allpublications cited herein are hereby incorporated by reference in theirentirety.

EXAMPLES Materials and Methods

Materials

HEPES-buffered Dulbecco's modified Eagle's medium (DMEM), Ham's/F12medium, trypsin-EDTA, fetal bovine serum (FBS), antibiotic-antimycoticsolutions, 0.05% trypsin-0.53 mM EDTA and anti-BrdU antibody werepurchased from Invitrogen (Carlsbad, Calif.). Hydrocortisone, dimethylsulfoxide (DMSO), insulin-transferrin-sodium selenite (ITSE) mediasupplement, mitomycin C (MMC), bovine serum albumin (BSA),5-bromo-2′-deoxyuridine (BrdU), Triton X-100, Hoechst 33258 dye,formalin, and Masson's Trichrome were all from Sigma-Aldrich (St. Louis,Mo.). Dispase II and epidermal growth factor (EGF) were obtained fromRoche (Indianapolis, Ind.). ΔNp63α polyclonal antibody and all thefluorescent dye-conjugated secondary antibodies were purchased fromBioLegend (San Diego, Calif.). Keratin-3 (clone AE5; CBL218) waspurchased from Millipore Corporation (Bedford, Mass.). Lgr6 antibody waspurchased from Santa Cruz Biotechnology (sc-48236, Santa Cruz, Calif.).

Native human PEDF (SEQ ID NO: 9) was purified from human plasma viacollagen I-sepharose resin as previously described by Petersen et al.(Pigment-epithelium-derived factor (PEDF) occurs at a physiologicallyrelevant concentration in human blood: purification andcharacterization. Biochem J 2003; 374(Pt 1):199-206), and was analyzedby SDS/PAGE and western blotting using an anti-PEDF antibody. Shortsynthetic peptides (39-mer, 34-mer, 29-mer, 25-mer, 20-mer, 18-mer, Mo29-mer, and Mo 20-mer) were synthesized and modified with acetylated atthe NH₂ termini and amidated at the COOH termini for stability andcharacterized by mass spectrometry (>95% purity) to order at GenScript(Piscataway, N.J.). In addition to SEQ ID NOs: 1-3, 5, and 7-8 describedhereinabove, two additional synthetic peptides were used in theexamples, in which the 25-mer has a sequence ofEQRTESIIHRALYYDLISSPDIHGT (SEQ ID NO: 4), whereas the 18-mer has asequence of EQRTESIIHRALYYDLIS (SEQ ID NO: 6).

Isolation and Culture of LSCs

LSCs were isolated from six-month-old New Zealand white rabbits and usedfor cell-suspension culture in accordance with the method previouslydescribed by Cheng et al. (The growth-promoting effect of KGF on limbalepithelial cells is mediated by upregulation of DeltaNp63alpha throughthe p38 pathway. J Cell Sci 2009; 122 (Pt 24): 4473-4480) and Wang etal. (Importin 13 serves as a potential marker for corneal epithelialprogenitor cells. Stem Cells 2009; 27: 2516-2526) with a slightmodification. Rabbit limbal tissues were washed in PBS containing 100U/ml penicillin and 50 μg/ml gentamicin. After carefully removing theiris, and excessive sclera, the limbal rings were exposed to dispase II(1.2 IU/ml in Hanks' balanced salt solution free of Mg²⁺ and Ca²⁺) at 4°C. for 16 hours. The loosened epithelial sheets were removed with a cellscraper and separated into single cells by enzymatic digestion (0.05%trypsin, and 0.01% EDTA) for 15 min at 37° C. with gentle shaking andtransferred to a stop medium (9 ml of HEPES-buffered DMEM containing 10%FBS). Cells were then collected by centrifugation (400×g for 5 min).

About 1×10⁵ cells were seeded on each wells of a 6-well plate andincubated in a DMEM/Ham's F-12 basal medium (10 mM HEPES, 5 ng/ml humanEGF, 1% SITE liquid medium, antibiotic-antimicotic solutions, 0.5% DMSOand 0.5 μg/ml hydrocortisone) supplemented with 10% FBS for 2 days, andthen shifted to a basal medium or basal medium containing 4.5 nM PEDF,25 nM 39-mer, 25 nM 34-mer, 25 nM 29-mer, 25 nM 25-mer, 25 nM 20-mer, 25nM 18-mer, 25 nM Mo 29-mer or 25-nM Mo 20-mer. The cells were continuedcultivated at 37° C. under 5% CO₂ for another 3 days until they reachedconfluence (passage 0). LSCs were co-cultured with MMC-treated NIH-3T3fibroblast feeder cells located within trans-well (0.4 μm pore, BDBiosciences, Bedford, Mass.). For passage purpose, near-confluent cellswere harvested again by enzymatic treatment (i.e., 0.25% trypsin), andthen 1×10⁵ subcultured cells were further cultured in the respectivemedium, and proliferative capacity of cells was compared every 4 daysafter continuously culture-subculture (passage).

Preparation of Feeder Cells

Confluent NIH-3T3 cells were incubated with 4 μg/ml MMC for 2 hours at37° C. under 5% CO₂, trypsinized and plated onto trans-well culturedishes (BD Biosciences) at a density of 2.2×10⁴ cells/cm². These feedercells were used 4 to 24 hours after plating.

Immunofluorescence Analysis

De-paraffinized tissue sections or 4% paraformaldehyde-fixed LSCs wereblocked with 10% goat serum and 5% BSA in PBST (PBS containing 0.1%Tween-20) for 1 hour. Staining was done using primary antibodies againstΔNp63α (1:150 dilution), BrdU (1:250 dilution), and keratin-3 (1:250dilution) at 37° C. for 2 hours, followed by incubation with theappropriate rhodamine- or FITC-conjugated donkey IgG (1:500 dilution)for 1 hour at RT. Nuclei were identified by counterstaining with Hoechst33258 for 7 minutes. Images were captured using a Zeiss epifluorescencemicroscope with a CCD camera and photographs were taken by using theAxiovert software.

BrdU Labeling

2×10⁵ limbal cells (the 1^(st) passage) were seeded at a slide coatedwith FNC COATING MIX® solution (Athena Enzyme Systems, Baltimore, Md.)and incubated with a culture medium for one day. BrdU (final, 10 μM) wasadded to the culture for 2 hours. After being fixed with 4%paraformaldehyde, cells were exposed to cold methanol for 2 minutes, andthen treated with 1 N HCl at RT for 1 hour before performingimmunofluorescence. For animal study, BrdU was reconstituted in DMSO asstock (80 mM). 10 μl of BrdU mixed with 90 μl of PBS wasintraperitoneally injected into mouse 3 hours prior to euthanasia. DNAsynthesis was assessed by BrdU labeling with anti-BrdU antibodies.

Corneal Wounding and Treatments

The experiments were performed using eight-week-old female C57BL/6 mice,and the procedures were approved by the Mackay Memorial Hospital ReviewBoard for animal investigation. Animals were anesthetized by anintraperitoneal injection of a mixture of zoletil (6 mg/kg) and xylazine(3 mg/kg). One filter paper (0.9 mm diameter) soaked with 20% ethanolwas placed on the central cornea of right eye for one minute and thenirrigated extensively with PBS. Subsequently, mechanical epithelialscrape was performed using a punch under a dissection microscope tocreate a circular injury (2 mm diameter) at the entire corneal region ofthe mouse eye without encroaching the corneal stroma, limbus orconjunctiva.

Each synthetic peptide (the 39-mer, 34-mer, 29-mer, 25-mer, 20-mer,18-mer, Mo 29-mer or Mo 20-mer) was reconstituted in DMSO as stock (5mM) and mixed with TOBREX® eye ointment (Alcon; containing 0.3%Tobramycin and 0.5% Chlorobutanol) to a concentration 50 μM. In each ofthe treatment group (n=6-10), the right eye was treated with 20 μl eyeointment containing the synthetic peptide once a day after the scrapeinjury. In the control group (n=20), right eye was treated with 20 μl ofeye ointment mixed with vehicle DMSO. Wound size was determined bystaining with topical fluorescein (Fluor-I-Strip, Ayerst Laboratories,Philadelphia, Pa.) and photographed with a digital camera. The area ofdefect was quantified from the photographs using a computer-assistedimage analyzer (Adobe Photoshop CS3 10.0) and was calculated as thepercentage of residual epithelial defect at each time point/initialwound area.

Cultivation of Hair Follicle Stem Cells

Human hair follicles were collected from hairs of healthy donors. Thefollicles were dissected with tweezers under microscope and most ofadipose and connective tissue was removed. The bulge region of hairfollicle under the sebaceous gland was cut and then bulge fragments(including central isthmus) were transferred into a 35-mm dishcontaining 1 ml of collagenase A (1 mg/ml; Roche) and incubated for 1.5hours at 37° C. to digest the collagen capsule. Subsequently, the bulgefragments were transferred into a fresh cell culture dish containingdispase II (2.4 U/ml)/0.05% trypsin and further incubated for 1.5 hoursat 37° C. to obtain a single cell suspension.

After mechanical dissection and enzymatic digestion, the suspended bulgecells isolated from five bulge fragments were seeded into a well of12-well culture plate and incubated with the basal medium for 5 days tofacilitate cell adhesion. The basal medium contains four parts Gibcocalcium-free Dulbecco's modified Eagle's medium (DMEM) and one partHam's F12 medium (calcium concentration reached about 0.25 mM) andsupplemented with 10% FBS, 10 ng/ml human epidermal growth factor, 500mg/I L-glutamine, 0.2% bovine pituitary extract, 0.18 g/mlhydrocortisone, 1% ITSE as well as antibiotic-antimicotic solutions.Cultivation method was similar to that of Blazejewska et al. (Stem Cells2009; 27:642). After 5 days of incubation, cells were cultured in theserum-free basal medium or basal medium containing 4.5 nM PEDF, 50 nM29-mer, or 50 nM 20-mer, until near confluence (about 2 weeks; passage0). The medium was changed every 2 to 3 days. The bulge cells wereco-cultured with 3T3 fibroblast feeder cells. For passage,near-confluent cells were harvested by enzymatic treatment (i.e., 0.25%trypsin) for 5 min at 37° C. Approximately 1×10⁵ human bulge cells weretransferred to a new dish and allowed to grow for 14 days to nearconfluence (passage 1).

Skin Wound Healing

To perform full-thickness skin excision, 8- to 10-week-old C57L/B6 micewere respectively anesthetized by an intraperitoneal injection of amixture of zoletil (6 mg/kg) and xylazine (3 mg/kg). Two full-thicknessskin excisional wounds, 4 mm in diameter were made on either side of thedorsal midline using Sklar Tru-Punch disposable biopsy punch (Sklar,West Chester, Pa., USA). In each treatment group (n=6), the skin woundwas treated with 25 μl skin ointment (containing 50 μM synthetic peptideand 0.006 μl DMSO vehicle) once daily after the skin scraping. In thecontrol group (n=6), skin wound was treated with 25 μl of skin ointmentwith equal concentration of DMSO. Each gram of the above skin ointmentcontains 5 mg neomycin sulfate and 12.5 mg Bacitracin Zinc. Wounds wereleft uncovered and harvested 4 and 7 days after injury. Mice were housedindividually during the healing period.

The area of wound was quantified from the photographs using acomputer-assisted image analyzer (Adobe Photoshop CS3 10.0) and wascalculated as the percentage of residual epithelial defect at each timepoint/initial wound area.

For histological analysis the complete wounds including 2 mm of theepithelial margins was isolated, bisected, fixed overnight in 4% PFA inPBS, and embedded in paraffin. Sections (5 μm) from the middle of thewound were stained using the Masson's trichrome procedure as describedby the manufacturer, in which the epithelium was stained red andconnective tissue was stained blue. Photographs were taken using a LeicaDC 500 camera (Leica Microsystems).

Statistics

Results were expressed as the mean±standard error of the mean (SEM).1-way ANOVA was used for statistical comparisons. P<0.05 was consideredsignificant, unless otherwise specified.

Example I

PEDF-Derived Short Synthetic Peptides Promote In Vitro LSC Proliferation

To identify the functional domain responsible for activating LSCproliferation, surface probability and hydrophilicity of the PEDFprotein (123 amino acids from positions 78-200 of the 418 amino acids ofhuman PEDF; GenBank number U29953) were analyzed and the amino acidsequence from positions 83-121 (referred as 39-mer) was identified to bethe most variable region. A serial of short peptides respectively coveramino acid sequence from residues 83-121 of PEDF, and variants thereofwere synthesized and then used to investigate their respective efficacyon proliferating LSCs in culture.

To determine the effects of PEDF-derived short peptides may have on theself-renewal of LSCs, LSC proliferation was examined by BrdUpulse-labeling (2 hours; red color) and LSC phenotype was revealed byimmunostaining ΔNp63α (green color).

As demonstrated in FIG. 1A, proliferation of LSCs cultivated in mediumcontaining full-length PEDF, 39-mer, 34-mer, 29-mer, or 20-mer was moresignificant than that in control medium (FIG. 1B; 22.0±1.5%, 30.1±1.9%,30.0±3.4%, 33.9±2.6% and 31.8±1.7% versus 3.0±0.5%). By contrast,enhanced proliferation was not observed in LSCs cultivated in a mediumcontaining the synthetic 25-mer and 18-mer PEDF.

Mo 29-mer and Mo 20-mer are derived from mouse PEDF, and they differfrom human 29-mer and human 20-mer by 4 and 2 amino acid residues,respectively. These mouse PEDF-derived short peptides displayed themitogenic activity to LSCs with an extent similar to their humancounterparts (FIG. 1B; 27.1±1.8% and 26.3±2.2%). Collectively, LSCexpansion in culture may be enhanced by medium supplemented with thefull-length PEDF, 39-mer, 34-mer, 29-mer, 20-mer, Mo 29-mer, orMo20-mer.

Example II PEDF-Derived Short Synthetic Peptides Promote In Vivo CornealWound-Healing

To investigate the effect of PEDF-derived short peptides on cornealwound healing in mice, corneal wound was created by a circular punch (2mm diameter), then control eye-ointment or eye-ointment containing theshort synthetic PEDF peptide(s) of this invention was applied to thewounded eye, and wound healing was subsequently evaluated by fluoresceinstaining. No significant differences in the size of the initial abrasionwere noted between synthetic PEDF peptide-treated and control mice.After 48 hours, complete corneal re-epithelialization was found in micetreated with either 39-mer, 34-mer, 29-mer, 20-mer, Mo 29-mer, or Mo20-mer, whereas control group mice and mice treated with 25-mer or18-mer exhibited incompletely healed wounds (FIG. 2A). 39-mer, 34-mer,29-mer, 20-mer, Mo 29-mer, or Mo 20-mer treatment resulted insignificantly smaller epithelial defect than that of the control mice at24 hours (37.2±4.3%, 40.6±7.2%, 36.5±6.4%, 42.2±4.3%, 33.8±5.2% and43.9±7.6% versus 84.9±5.1%) and 48 hours (4.8±2.7%, 4.6±2.1%, 3.7±2.9%,5.0±3.7%, 4.6±2.3%, and 6.2±3.5% versus 39.5±3.1%) (FIG. 2B).

Example III PEDF-Derived Short Synthetic Peptides Promotes In Vivo LSCProliferation after Corneal Wounded

To investigate whether the LSC proliferation during corneal woundhealing could be accelerated by the synthetic peptide of this invention,mice were intraperitoneally received BrdU and euthanized at 24 hoursafter corneal wounding. ΔNp63α (green) and BrdU (red)double-immunostaining of ocular sections revealed that the levels ofBrdU- and ΔNp63α-positive cells in the limbus of 29-mer and 20-mertreated eyes were elevated, as compared with that of the control eyes(FIGS. 3A and 3B; 43.2±4.6% and 42.4±4.3% versus 15.0±7.6%); whereas thelevel of LSC proliferation in 18-mer treated eyes was similar to that ofthe control animals.

On day 5 post-wounding, HE stain and ΔNp63α (green) immunostaining ofocular sections revealed that the thickness of the limbus of 29-mer,20-mer and Mo 20-mer-treated eyes returned to full-thickness ofstratified layers, as compared with that of the normal unwounded eye;whereas the limbal epithelia of 18-mer treated- or controlointment-treated eyes were thinner, and with fewer ΔNp63α-positive LSCspresent (FIG. 4A). On average, the levels of ΔNp63α-positive LSCs inlimbus of normal unwounded eye is 74.8±6.3%; whereas the levels ofΔNp63α-positive LSCs in corneal wounded eyes treated with control,29-mer, 20-mer, 18-mer and Mo 20-mer ointment were 38.9±2.8%, 77.0±4.1%,73.1±8.6%, 39.4±2.6%, and 76.3±6.2% respectively (FIG. 4B). Takentogether, 29-mer or 20-mer treatment can activate LSCs in vivocoincidently with more prominent corneal re-epithelialization thancorneal regeneration by native progress.

The results from the preceding examples establish that present syntheticPEDF peptides (such as the 29-mer, 20-mer, Mo 29-mer, and Mo 20-mer) mayenhance proliferation while suppresses spontaneous differentiation ofLSCs in cultures. This may significantly improve the quality of limbalequivalent and is therefore advantageous to the corneal regenerativemedicine. The direct stimulation of the proliferation of limbalprogenitor cells and the ensued acceleration of cornealre-epithelialization suggest that the present synthetic PEDF peptidesmay act as potential agents to treat LSC deficiency-related disorders.

Example IV PEDF-Derived Short Synthetic Peptides Promote In VitroProliferation of HFSCs

The effects of full-length PEDF and its short peptides on the HFSCproliferation was investigated in this Example using BrdU pulse-labeling(2 hours; red color), and HFSC phenotype was revealed by immunostainingLgr6 (green color). As demonstrated in FIG. 5A, proliferation of humanHFSC cultivated in medium containing 20-mer was more significant thanthose cultivated in control medium. FIG. 5B illustrated that for thegroups treated with full-length PEDF, 29-mer and 20-mer, the percentageof BrdU and Lgr6-double positive cells per total Lgr6-positive cells is73.8±6.0%, 86.2±3.9% and 79.5±2.6%, in contrast to 31.9±5.6% and30.1±6.3% of the control and 18-mer-treated groups.

Example V PEDF-Derived Short Synthetic Peptides Accelerate EpithelialWound-Healing

Referring to FIG. 6A, groups treated with 39-mer, 34-mer, 29-mer,20-mer, Mo 29-mer and Mo 20-mer showed enhanced wound healing on day 4,post-injury. The residual epithelial defects of these groups on day 4were 48.1±3.2%, 41.4±2.5%, 46.3±3.7%, 50.1±3.8%, 47.8±4.3%, and49.6±3.9%, respectively; as compared to 61.5±3.2% of the control group(FIG. 6B). At day 7 post-injury, the wounds in animals treated withthese peptides respectively exhibited near complete healed wounds, andscars were less observed (FIG. 6A). The residual epithelial defects inthese groups at day 7 were 18.4±5.7%, 17.9±6.1%, 18.2±4.3%, 20.0±4.9%,16.8±6.6% and 20.3±7.5%, respectively; as compared to the control groupof 44.5±5.3% (FIG. 6B). However, 25-mer and 18-mer were not able topromote wound healing (FIGS. 6A and 6B). The results indicated thatPEDF-derived short peptides according to the present disclosure (e.g.,39-mer, 34-mer, 29-mer, 20-mer, Mo 29-mer, and Mo 20-mer) benefit skinre-epithelialization.

To further confirm the efficacy of 29-mer and 20-mer on skin woundrepair, skin sections from day 4 after trauma were stained using theMasson's trichrome. Referring to FIG. 7A, it is evident that animalstreated with 29-mer and 20-mer exhibited better wound healing thancontrol group. The residual epithelial defect of 29-mer and20-mer-treated groups were 52.7±6.2% and 49.7±5.6%, respectively; ascompared to control animals of 63.8±5.8% (FIG. 7B). In addition, 29-mertreatment elevated the thicknesses of hyper-proliferative epithelial(HE) tissue and granulation tissue (GT) around the edge of the wound(FIG. 7C). Quantitative results of the area of HE tissue indicated thatthe skin thickness in 29-mer and 20-mer treated groups were 1.41±0.25and 1.32±0.21 folds over that of the control group, respectively (FIG.7D). In addition, quantitative results of the area of GT indicated thatthe skin thickness in 29-mer and 20-mer treated animals wererespectively 1.45±0.23 and 1.37±0.17 folds more than those of thecontrol group (FIG. 7E).

Masson's trichrome was used to stain skin sections obtained from day 7after trauma for the measurement of the wound closure. 29-mer, Mo29-mer, or 20-mer-treated wounds exhibited better re-epithelialization,in which the smaller residual epithelial defects was identified thanthat of the control ointment-treated wound (FIGS. 8A and 8B; 17.9±3.3%,17.2±7.3%, and 20.8±6.1% versus 43.5±6.5%).

Example VI PEDF-Derived Short Synthetic Peptides Accelerate EpithelialWound-Healing by Promoting Proliferation in the Basal Cells of theHyper-Proliferative Epithelium

To investigate whether skin resurfacing process, which involves cellreplication, is accelerated by 29-mer, Mo 29-mer, 20-mer, or Mo 20-mer,wounds were treated with skin ointment containing 29-mer, Mo 29-mer,20-mer, or Mo 20-mer for 4 days, and then mice were intraperitoneallyinjected with BrdU for further 3 hours before euthanized.Immunohistochemical analysis of skin specimens using the anti-BrdUantibody revealed that the distributions of BrdU-positive cells areprincipally located at the basal layer of HE tissue and the bulge regionof hair follicle (FIG. 9A). The numbers of BrdU-positive cells in29-mer, Mo 29-mer, 20-mer, and Mo 20-mer-treated wounds increasedsignificantly; as compared with that of the control wound (FIG. 9B;47.9±5.0%, 52.5±2.5%, 53.1±6.5% and 49.2±4.3% versus 30.8±8.1%). Thisobservation was consistent with the thicker HE tissue treated with29-mer or 20-mer.

Example VII PEDF-Derived Short Synthetic Peptides Promotes HFSCProliferation after Skin being Wounded

Lgr6-positive HFSCs is an important precursor cells for epitheliumrepair during skin wound healing. As demonstrated in FIG. 10A,immunohistochemical analysis of skin specimens obtained from woundstreated with 20-mer for 4 days revealed that the Lgr6-positive cellswere markedly increased at HE tissue, as compared with that of thecontrol wound. Immunohistochemical studies also revealed thatLgr6-positive cells were partly located at the basal layer of HE tissue.This observation is consistent with the finding that cell proliferationmainly occurred at basal layer of HE tissue. Dual-immunostaining furtherconfirmed that Lgr6-positive basal cells (green) are responsible for theenhanced proliferation found in HE tissue induced by the synthetic20-mer (FIG. 10B).

The results from the preceding examples establish that present syntheticPEDF peptides (such as the 20-mer, 29-mer, 34-mer, 39-mer, Mo 20-mer,and Mo 29-mer) may enhance proliferation of HFSCs in cultures.Specifically, the enhanced proliferation of HFSCs is associated withre-epithelialization and wound-healing. Accordingly, the presentsynthetic PEDF peptides are suitable for use as a therapeutic agent topromote wound-healing, especially for healing wounds with large area orwounds that are difficult to heal due to diabetes or aging.

It will be understood that the above description of embodiments is givenby way of example only and that various modifications may be made bythose with ordinary skill in the art. The above specification, examples,and data provide a complete description of the structure and use ofexemplary embodiments of the invention. Although various embodiments ofthe invention have been described above with a certain degree ofparticularity, or with reference to one or more individual embodiments,those with ordinary skill in the art could make numerous alterations tothe disclosed embodiments without departing from the spirit or scope ofthis invention.

What is claimed is:
 1. A synthetic peptide for promoting the proliferation of limbal epithelial stem cells or hair follicle stem cells, consisting of an amino acid sequence having 20-39 amino acid residues in length and at least 80% amino acid sequence identity to SEQ ID NO: 1, wherein the amino acid sequence comprises at least 20 consecutive residues identical to residues 11-30 of SEQ ID NO:
 1. 2. The synthetic peptide of claim 1, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:
 5. 3. A composition for promoting the proliferation of limbal epithelial stem cells or hair follicle stem cells, comprising, an effective amount of the synthetic peptide of claim 1, and a carrier.
 4. The composition of claim 3, wherein the carrier is a powdered culture medium suitable for culturing the stem cells.
 5. The composition of claim 4, wherein the synthetic peptide is present in an amount of 1-100 nM.
 6. The composition of claim 5, wherein the synthetic peptide is present in an amount of 25-50 nM.
 7. The composition of claim 3, wherein the carrier is a pharmaceutically acceptable carrier selected from the group consisting of a liquid, gel, cream, ointment, adhesive, amniotic membrane, skin substitute, artificial skin, and skin equivalents.
 8. The composition of claim 3, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:
 5. 